2′ Modified oligonucleotides

ABSTRACT

Oligomers which have substituents on the 2′ position are resistant to oligonucleases and furthermore can be derivatized to deliver reagents or drugs, to carry label, or to provide other properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/131,647,filed Aug. 10, 1998, now U.S. Pat. No. 6,476,205, which is acontinuation of application Ser. No. 08/467,422, filed Jun. 6, 1995, nowU.S. Pat. No. 5,792,847, which is a continuation of application Ser. No.08/240,508, filed May 10, 1994, now U.S. Pat. No. 5,466,786, which is acontinuation of application Ser. No. 07/425,857, filed Oct. 24, 1989,now abandoned. The disclosure of each of the foregoing is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

The invention relates to modified oligo-nucleotides useful intechnologies which rely on complementarity or specificity of oligomersequences for drug delivery or for direct interference with nucleic acidactivity. More specifically, the invention concerns oligomersderivatized at the 2′ position, which are stable to nuclease activity.

BACKGROUND ART

There has been considerable activity in recent years concerning thedesign of nucleic acids as diagnostic and therapeutic tools. One aspectof this design relies on the specific attraction of certain oligomersequences for nucleic acid materials in vivo which mediate disease ortumors. This general approach has often been referred to as “anti-sense”technology. An oversimplified statement of the general premise is thatthe administered oligomer is complementary to the DNA or RNA which isassociated with, and critical to, the propagation of an infectiousorganism or a cellular condition such as malignancy. The premise is thatthe complementarity will permit binding of the oligomer to the targetnucleic acid, thus inactivating it from whatever its ordinary functionmight have been.

A simple illustration would be the administration of a DNA oligomercomplementary to an mRNA which encodes a protein necessary to theprogress of infection. This administered DNA would inactivate thetranslation of the mRNA and thus prevent the formation of the protein.Presumably the DNA could be directly administered, or could be used togenerate an mRNA complement to the target mRNA in situ. There is by nowextensive literature concerned with this general approach, and themethods of utilizing oligomers of this type which are complementary totarget RNA or DNA sequences are set forth, for example, in van der Krol,A. R., et al., Biotechniques (1988) 6:958-976; Stein, C. A., et al.,Cancer Research (1988) 48:2659-2668; Izant, J. G., et al., Science(1985) 229:345-352; and Zon, G., Pharmaceutical Research (1988)5:539-549, all incorporated herein by reference. In addition, abibliography of citations relating to anti-sense oligonucleotides hasbeen prepared by Dr. Leo Lee at the Frederick Cancer Research Facilityin Frederick, Md.

There are two conceptual additions to the general idea of usingcomplementarity to interfere with nucleic acid functionality in vitro.The first of these is that strict complementarity in the classicalbase-pairing sense can be supplemented by the specific ability ofcertain oligonucleotide sequences to recognize and bind sequences indouble-helical DNA and to insert itself into the major groove of thiscomplex. A fairly recent but reasonably definitive series of papers haselucidated the current rules for such specificity. These papers takeaccount of very early work by, for example, Arnott, S., et al., J MolBiol (1974) 88:509-521, which indicates the general principle of bindingas triplexes poly-dT/poly-dA/poly-dT, and the corresponding analogoustriplex involving poly-dC as summarized by Moser, H. E., et al., Science(1987) 238:645-650. More recent studies show that the earlier rule(which was that recognition could be achieved by a homopyrimidineoligomer to homopurine/homopyrimidine stretches in the duplex) could beextended to patterns whereby mixed sequences can also be recognized(Griffin, L. C., et al., Science (1989) 245:907-971. Further summariesof these phenomena are given, for example, in a review article by MaherIII, L. J., et al., Science (1989) 245:725-730. Additional relateddisclosures of triple-helix formation are those by Cooney, M., et al.,Science (1988) 241:456-459; Francois, J. -C., Nucleic Acids Res (1988)16:11431-11440; and Strobel, S. A., et al., J Am Chem Soc (1988)110:7927-7929. While further details are needed to provide exactsequence specificity studies in this context, it is clear that the rulesfor “complementarity” in this sense of specific embedding into the majorgroove of the double-helix are rapidly emerging.

The second aspect of anti-sense technology which deviates from thesimple concept of base-pair complementarity in native oligonucleotidesresults from the early recognition that oligonucleotides, especiallyRNAS, are highly susceptible to nuclease cleavage in biological systems.In order for these materials to remain active drugs, it would benecessary to stabilize the administered oligonucleotides against thisdegradation. The approach that has so far been used has been to modifythe phosphodiester linkages so as to be resistant to attack by theseenzymes. In particular, the phosphodiester linkage has been replaced byphosphoramidate linkages, methylphosphonate linkages, andphosphorothioate linkages. These approaches have certain results withregard to stereoisomerism and its associated impact on hybridization tothe target sequences that make them less than completely satisfactory.An alternate approach has been to modify the nucleosides by using2′-O-methyl ribose or the alpha-anomers of the conventional nucleosideresidues. In addition, oligomers containing 2′ amino groups have beenprepared via their triphosphate analogs and enzyme-catalyzedpolymerization by Hobbs, J., et al., Bio-chemistry (1973) 12:5138-5145.Some of these approaches have been summarized in the Zon review cited inthe previous paragraph.

The present invention provides additional 2′-substituted pentosemoieties for inclusion in the oligomers useful in this technology whichare resistant to nuclease activity, and may optionally be combined withadditional modifications such as those set forth above.

DISCLOSURE OF THE INVENTION

The invention is directed to nucleosides and nucleotides of the formula:

wherein

B is a purine or pyrimidine residue or analog thereof;

W₁ is H, (PO₃)_(m) ⁻² wherein m is an integer of 1-3; a protectinggroup, or a group reactive to link hydroxyl groups;

W₂ is H, PO₃ ⁻², a protecting group, or a group reactive to linkhydroxyl groups;

X is O, S, NR or CR₂ wherein each R is independently H or alkyl (1-6 C);

Y is a linker moiety, a drug residue optionally attached through alinker moiety, a label optionally attached through a linker moiety, or aproperty-affecting residue optionally attached through a linker moiety,wherein said X-Y substituent renders an oligomer in which saidnucleoside or nucleotide of formula (1) is included more stable totreatment with nuclease than said oligomer which incorporates acorresponding nucleotide having —H₂ or —HOH at the 2-position.

These materials are useful as intermediates in the synthesis of theoligomers of the invention, which are oligomers of the formula:

wherein each B is independently a purine or pyrimidine residue or analogthereof;

W₃ and W₄ are each independently H, PO₃ ⁻², a protecting group, or agroup reactive to link hydroxyl groups;

n is an integer of 1-200;

each Z is independently a nucleotide linking residue covalentlyconjugating the hydroxyl groups of sequential nucleotide residues;

each A is independently selected from the group consisting of H, OH, OHderivatized to a protecting group, and X-Y wherein

X is O, S, NR, or CR₂ wherein each R is independently H or alkyl (1-6C); and

Y is a linker moiety, a drug residue optionally attached through alinker moiety, a label optionally attached through a linker moiety, or aproperty-affecting group optionally attached through a linker moiety;

wherein at least one A is X-Y; and

wherein the oligomer is more stable to nuclease than the correspondingoligomer wherein all A are H or OH.

The oligomeric materials of formula (2) are useful as therapeutic orprophylactic agents in protocols which are directed against infectiousdisease or malignancy by targeting specific DNA and/or RNA sequencesassociated with the condition, as well as in diagnostic applications.

MODES OF CARRYING OUT THE INVENTION

A. Definitions

The oligomers of the invention contain the residue of at least onenucleotide of formula (1). In this formula, and in the oligomers, Brepresents a conventional purine or pyrimidine base such as adenine (A),thymine (T), cytosine (C), guanine (G), or uracil (U) or protected formsthereof. Suitable protecting groups include acyl, isobutyryl, benzoyl,and the like. B can, however, also represent a modified or protectedform or related derivative of these conventionally occurring bases,i.e., an “analog.” A wide range of these analogous heterocyclic bases isknown in the art. For example, commonly encountered among such analogsare those which include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thioridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil,5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyaceticacid methylester, uracil-5-oxyacetic acid (v), wybutoxosine,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,3-(3-amino-3-N-2-carboxypropyl)uracil,(acp3)w, and 2,6-diaminopurine.

As the modification of these bases affects both the stability of theresulting oligomer and the hybridization ability of the sequence, inmany instances only a limited number of such substitutions in aparticular oligomer is desirable. However, there are other instanceswhen an entire oligomer may be composed of nucleotide residuescontaining an analog. For example, DNA polymers of uridine and oligomersof 5-bromouridine, and 5-methyl cytidine have been shown to betherapeutically and diagnostically useful. As will be apparent topractitioners of the art, a sensible approach must be used in designingoligomers containing either conventional or modified base forms so thatthe properties of the resulting monomer are in the desired range.Therefore, in some cases, less than 10% of the bases indicated as “B” inthe sequence of formula (2) will be replaced by analogous bases,preferably less than 5%, more preferably less than 1%, and mostpreferably none at all. However, in other cases, complete replacement byanalogs is desirable.

Similar comments apply to the substitution for the bases ofnonfunctional substituents such as alkyl or aryl nonheterocyclic groups;however, such substitutions may be permissible to a highly limitedextent, for example one residue per 20 or so without actually destroyingthe functionality of the oligomer. It does not appear there is anyparticular advantage in making these replacements, and they arepermissible only because the replacement may be overwhelmed by thefunctionality of the remainder of the molecule.

Substituents designated W₁-W₄ may be H, PO₃ ⁻², (PO₃)_(m) ⁻² ,protecting groups, or groups reactive to link hydroxyl group.

A “protecting group” in the context of W is a substituent which preventsthe reactivity of the —OH to which it is bound in a chemical reaction,typically a reaction to link sequential nucleotides, and which can beremoved when the reaction is completed. Typical protecting groups in thecompounds of the invention include 4,4′-dimethoxy trityl (DMT),4-monomethoxytrityl and trityl.

A “group reactive to link hydroxyls”, is an intermediate residue in theformation of an internucleotide link between the 5′ and 3′ hydroxyls.Thus, the reaction of said group with the appropriate —OH of theadjacent nucleotide results in the nucleotide linking residue, Z.

The linking residue represented by Z is typically P(O)O⁻ in naturallyoccurring oligonucleotides, but can also be P(O)S, P(O)NR₂, P(O)R, orP(O)OR′, or can be CO or CNR₂ wherein R is H or alkyl (1-6C) and R′ isalkyl (1-6C) or can be —CX₂— wherein each X is independently anelectron-withdrawing substituent as described in copending applicationattorney docket 4610-0005, filed on even date herewith, assigned to thesame assignee and incorporated herein by reference. In general, Z can beany nucleotide linking moiety conventionally used to conjugatenucleotide residues to form oligonucleotides.

A linker moiety, as represented by Y (Y′ when covalently bound to anadditional substituent), is any bivalent bridging residue used to attacha desired substituent to the monomer or oligomer. The linker may besimply binding methylene groups—i.e., —(CH₂)_(n)— or may includeheteroatoms and functional groups, e.g., —CH₂OCH₂CH₂O— or—CH₂O—CH₂CH₂NH— or —COOCH₂CH₂O—. The linker residue may also be aresidue derived from a commercially available bifunctional linker suchas the hetero- and homo-bifunctional linkers marketed by Pierce ChemicalCo., Rockford, Ill.

A “drug residue,” represented by Y, is the attached portion of a druguseful in conjunction with the oligomer, such as a drug capable ofintercalation or of insertion into the minor groove of a DNA-DNA orDNA-RNA double helix or which can effect oligonucleotide cleavage.Examples of such drugs are set forth hereinbelow.

A “label residue” is the attached portion of a label such as a moietycontaining a radioisotope, a fluorophore, a chromophore, an enzyme andthe like. Such labels may be desirable if this oligomer is to be used indiagnosis.

A “property-affecting” residue is a residue which, by virtue of itspresence, results in changed properties of the oligomer. Such changedproperties include, but are not limited to, enhancement of cellpermeation properties, enhancement of the ability of the oligomer tohybridize to or otherwise bind to oligonucleotide sequences andenhancement of stability to nucleases.

Compounds of the invention that contain groups which are negativelycharged at neutral pH can be prepared as their salts. The salts areformed from inorganic bases, such as NaOH, KOH or Ca(OH)₂, or organicbases, such as caffeine, various alkylamines, TEA, and DBU.

Compounds of the invention that contain groups which are positivelycharged at neutral pH can be prepared as acid-addition salts formed frominorganic acids such as HCl, H₂SO₄ or H₃SO₄, or from organic acids suchas acetic, succinic or citric.

Thus, the nucleotides and their corresponding oligonucleotides may existas salts depending on the pH at which they find themselves or at whichthey are prepared. The phosphate and phosphodiester moieties associatedwith these molecules permits the formation of basic salts such as thoseformed from inorganic ions such as sodium, potassium, ammonium ions, butespecially divalent ions such as calcium and magnesium ions. It ispossible, but less common, to form salts of these materials with organicbases such as organic amines or heterocycles.

The invention compounds differ from those of the prior art by having inthe 2′ position, at either chirality, a substituent which confersnuclease stability and, optionally, provides the capacity to deliver adrug, for example, a reagent which is effective to interact with duplexDNA in its minor groove, provides a label, or provides some additionalproperty. While the remainder of the molecule in formula (2) issometimes shown for convenience as having the features of the nativeoligonucleotides, and, indeed, this is often the most preferredembodiment, also included within the invention are molecules whichcontain the 2′ extensions and substitutions of the invention, but alsocontain additional modifications such as replacement of one or more ofthe phosphodiester linkages with, for example, a phosphorothioate ormethyl phosphonate linkage; a phosphoramidate linkage, including thosecontaining organic amino substituents, such as morpholidates,replacement of all beta-anomers by the alpha-anomer, and the presence orabsence of protecting groups or phosphate residues at the 5′- and3′-termini.

At the 2′ position, the invention discloses several general categoriesof substituents, which share a common type of linkage to the 2′ carbonthrough a substituent selected from O, S, R and CR₂, wherein each R isindependently selected. In all embodiments, X-Y represents a substituentwhich is capable, by virtue of its presence, of inhibiting the cleavageof the oligomer in which it is included by nucleases. All of theoligomers of the invention are relatively stable to nucleases.

The stability of the oligomers to nucleases can be determined using anyconvenient assay, but is conveniently assessed using the snake venomassay illustrated hereinbelow. This assay is conducted as follows: Theassay buffer is 0.5 M Tris HCl, pH 8.0, containing 100 uM/MgCl₂.Commercially available phosphodiesterase isolated from Croatalusdurissus is obtained from Boehringer Mannheim as a 50% (v/v) solution inglycerol, pH 6, with a specific activity of approximately 1.2 U/mg. Oneul of the phosphodiesterase-containing solution is added to 100 ulbuffer, and oligomers are tested by reconstituting 0.15 OD of oligomerin the 100 ul buffer/venom prepared above. Degradation is monitored byobserving the disappearance of the 260 nm absorption of the oligomer atits characteristic retention time on HPLC, and measuring the appearanceof degradation products.

The oligomers of the invention which contain at least one nucleotideresidue containing the 2′ substituent are more stable to nuclease asjudged by the foregoing assay than the corresponding oligomer containingan unsubstituted 2′ position in place of the substituted positions inthe invention compounds. By comparing the rate of hydrolysis in thesnake venom assay with the invention compound, with that of thecorresponding oligomer which is not derivatized in the 2′ position, itcan be assessed whether the presence of the 2′ substituent(s) stabilizedthe oligomer to cleavage by nucleases.

Typical embodiments of Y, when its sole function is to alter theproperties of the oligomer, include alkyl or alkenyl (2-20C), preferably2-6C, which may or may not be substituted with noninterferingsubstituents, such as —OH, ═O, carboxyl, halo, amino groups and thelike, aryl or substituted aryl (6-20C), various alkyl silyl derivativesof the formula SiR₃ (wherein each R is alkyl of 2-6C), and similarsubstituents which also contain heteroatoms. If Y includes a linkermoiety, this portion of Y (Y′) will provide functional group(s) forconjugation to additional substances. For example, an embodiment of Y′,of 1-20C may contain a hydroxyl, amino, mercaptyl, carboxy, keto, orother functional group or several of these in combination. Typicalexamples include —CH₂COOH; —CH₂CONH₂; —CH₂COOEt; —CH₂CONHCH₂CH₂NH₂; andthe like.

The linker moiety may be utilized to couple the nucleoside, nucleotideor residue within the oligomer to a reagent or drug, such as a drugwhich is known to interact with the minor groove of duplex DNA orDNA/RNA. A wide variety of these reagents and substances is known andthe function, in vivo, is generally to inactivate the DNA duplex withwhich these reagents interact. Typical examples of such agents includenetropsin and its derivatives, anthramycin, quinoxaline antibiotics,actinomycin, pyrrolo (1-4) benzodiazepine derivatives and intercalatingagents.

Other drugs, besides those which seek the minor groove, may also beused. Intercalators, toxins, degradation inducers, and the like can alsobe used. Furthermore, the drug need not be linked through the linkermoiety, but may be directly associated with the substituent X, dependingon the chemistry of the particular drug.

Another embodiment of Y represents label optionally linked to X througha linker moiety, but also possibly directly attached, again depending onthe chemistry in the particular case. Suitable labels includeradio-isotopes, fluorescent labels, such as fluoroscein and dansyl,chromophores, enzymes and the like. A wide variety of labels is knownand can be used to provide detectability when the oligomers of theinvention are used as probes or in other specific binding diagnosticassays.

Finally, Y can be a substituent which confers altered properties on theoligomer. It has already been noted that all of the substituents,including drugs and label, confer increased nuclease stability. However,additional properties may also be affected—for example, agents whichcleave associated nucleotide chains may be attached; cell permeationenhancement may occur by virtue of the substituent, or Y may enhance thehybridization of the oligomer to complementary oligonucleotides or to aDNA/DNA or DNA/RNA helix. As with all of the foregoing embodiments of Y,the activity or property-changing substituent may be directly bound to Xor may be conjugated through a linker moiety.

B. Preparation of the Invention Compounds

Some of the compounds of the invention which are nucleosides ornucleotides are prepared by reacting the corresponding nucleotide ornucleoside having OH in the 2′ position with suitable reagents to effectconversion to the substituted form. In some cases, the 2′ substituentmay be derived from cyclic forms of the nucleoside or nucleotide.Further conversions may be required, as illustrated below, to activatethe nucleotide or nucleoside for inclusion into the oligomer. Thetechniques for these conversions are generally understood in the art, asare the techniques for sequential synthesis of the oligomers.

In particular, dimers may be synthesized to evaluate the effect of the2′ substituent on nuclease activity. In the formation of the dimer, theconverted nucleoside or nucleotide of the invention, protected at the 5′position and containing a group reactive to link hydroxyl groups at the3′ position, is reacted with, for example, thymidine or cytidine linkedat the 3′ position to solid support and the resulting dimer is cleavedfrom the support and deprotected.

In all of the foregoing cases, conversions to change the functionalityand character of the 2′ substituent can be conducted either at themonomer or oligomer level. Thus, a 2′ substituent which has the formulafor X-Y OCH₂COOEt can be converted to an embodiment wherein thesubstituent is the free acid or the amide either when the ester is asubstituent of the single nucleoside or nucleotide, of a diner, orcontained in the oligomeric chain.

The compounds of the invention which are oligomers are obtained byinclusion of the derivatized nucleotide or nucleoside into the oligomerusing standard solid phase oligonucleotide synthesis techniques. Suchtechniques are commercially available for formation of both standardphosphodiester linkages and the conventional substitute linkagesdescribed above.

C. Utility and Administration

The compounds of the invention are useful in a manner known in the artfor nuclease-inhibited, specifically complementary or binding,oligomers. As set forth above, the general methods for utilization ofthese compounds are known, and their application to specific diseases orconditions depends on the ascertainment of the appropriate bindingspecificity. The determination of this binding specificity does notaffect the manner of preparation or application of the modifiedcompounds of the invention.

Accordingly, the modified oligomers of the invention are useful intherapeutic, diagnostic and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general—as described above, antisense therapy asused herein includes targeting a specific DNA or RNA sequence throughcomplementarity or through any other specific binding means, forexample, sequence-specific orientation in the major groove of the DNAdouble-helix, or any other specific binding mode. For such therapy, theoligomers of the invention can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,latest edition.

For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the oligomers of the invention are formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the oligomers may beformulated in solid form and redissolved or suspended immediately priorto use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives. In addition,detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays, for example, or usingsuppositories. For oral administration, the oligomers are formulatedinto conventional oral administration forms such as capsules, tablets,and tonics.

For topical administration, the oligomers of the invention areformulated into ointments, salves, gels, or creams, as is generallyknown in the art.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are conducted by hybridization through basecomplementarity or triple helix formation which is then detected byconventional means. For example, the oligomers may be labeled usingradioactive, fluorescent, or chromogenic labels and the presence oflabel bound to solid support detected. Alternatively, the presence of adouble or triple helix may be detected by antibodies which specificallyrecognize these forms. Means for conducting assays using such oligomersas probes are generally known.

In addition to the foregoing uses, the ability of the oligomers toinhibit gene expression can be verified in in vitro systems by measuringthe levels of expression in recombinant systems.

It may be commented that the mechanism by which the specifically-bindingoligomers of the invention interfere with or inhibit the activity of atarget RNA or DNA is not always established, and is not a part of theinvention. If the oligomer seeks, for example, a target mRNA,translation may be inhibited. In addition, by binding the target, thedegradation of the mRNA message may be enhanced, or the furtherprocessing of the RNA may be inhibited. By formation of a triple helix,the transcription or replication of the subject DNA may be inhibited;furthermore, reverse transcription of infectious RNA or replication ofinfectious DNA is interfered with. It is also thought that the immunefunction may be modulated through physiological mechanisms similar tothose induced by double-stranded RNA as exemplified by the “ampligen”system or similar to those used to suppress systemic lupuserythematosus. The oligomers of the invention are characterized by theirability to target specific oligonucleotide sequences regardless of themechanisms of targeting or the mechanism of the effect thereof.

Finally, it is understood that the oligonucleotide can be derivatized toa variety of moieties which include, intercalators, chelators,lipophilic groups, label, or any other substituent which modifies butdoes not materially destroy the oligomeric character of the backbone.

The following examples are intended to illustrate but not to limit theinvention. In all synthesis reactions, a dry argon atmosphere was used.

EXAMPLE 1 Preparation of Nucleotides and Oligomers (A=NHAc)

2′-N-acetaminouridine was protected at the 5′-position and made reactiveat the 3′-position for formation of a phosphodiester linkage conjugatingthe residues in an oligomer as follows:

2′-N-Acylaminouridine. To 152 mg of 2′-N-acylamino-3′,5′-O-diacyluridine (Verheyden, J. P. H, et al., Org Chem (1971)36:250-254) (0.411 mmol) in 25 ml of MeOH was added a catalytic amountof KCN. After 15 h, 1.00 g of silica gel was added, and the reaction wasconcentrated. The powder was added to the top of a 20 mm column ofsilica gel that had been equilibrated in 5% H₂O in CH₃CN. The column waseluted with 5% H₂O in CH3CN using standard flash chromatographyconditions (Still, W. C., et al., J Org Chem (1978) 43:2923-2925).Isolation and concentration of the product afforded 59.5 mg (50.8%yield) of product.

2′-N-Acylamino-5′-O-(4,4′-dimethoxytrityl)-uridine. To 59.5 mg of2′-N-acylaminouridine (0.208 mmol) in 2.5 ml of dry pyridine (that wasfirst concentrated from dry pyridine) was added 77.6 mg (0.229 mmol,1.10 equiv) of 4,4′-dimethoxytritylchloride. The reaction was stirred aroom temperature for 15 h and then diluted with 3.0 ml of H₂O. Themixture was partitioned between H₂O and Et₂O, shaken and separated. Theaqueous layer was extracted with Et₂O, and the combined organics werewashed with 1% aqueous NaHCO₃, dried (Na₂SO₄), filtered, andconcentrated. The residue was purified by flash chromatography on a 20mm column using first one column volume of CH₂Cl₂ and then 8% MeOH inCH₂Cl₂ as eluants. Isolation and concentration afforded 70.7 mg ofproduct (56.6% yield) as a colorless foam.

2′-N-Acylamino-5′-O-(4,4′-dimethoxytrityl)-uridin-3-yl-hydrogenphosphonatetriethylammoniumsalt. To a mixture of 132 mg of 1,2,4-triazole (1.91 mmol) and 0.476 mlof anhydrous 4-methylmorpholine (4.33 mmol) in 2.40 ml of dry CH₂Cl₂ wasadded 0.236 ml of a 2.0 M solution of PCl₃ in CH₂Cl₂ (0.472 mmol). Themixture was then cooled on an ice-water bath for 30 min. To this mixturewas added a solution of 70.7 mg of2′-N-acylamino-5′-O-(4,4′-dimethoxytrityl)-uridine (0.188 mmol,previously concentrated from dry pyridine) in 0.523 ml of dry pyridine,dropwise over several minutes. The reaction was stirred for 20 min andthen poured onto 16.8 ml of cold 1 M aqueous triethylammoniumbicarbonate (TEAB,pH=9.0). The mixture was rapidly stirred for 15 minand then extracted with 2×17 ml of CH₂Cl₂. The combined organics werewashed with 11.7 ml of 1 M aqueous TEAB, dried (Na₂SO₄), filtered, andconcentrated. The residue was purified by flash chromatography on a 20mm column using one column volume of 1% TEA in CH₂Cl₂, then one columnvolume of 1% TEA and 2.5% MeOH in CH₂Cl₂,and then 1% TEA and 10% MeOH inCH₂Cl₂. The product was isolated and concentrated. The residue waspartitioned between CH₂Cl₂ and 1 M aqueous TEAB, shaken and separated.The organic layer was dried (Na₂SO₄), filtered and concentrated. Theproduct was concentrated from dry CH₃CN affording 41.9 mg (43.6% yield)of product as a slightly yellow foam.

The resulting title compound is coupled into oligomers using the methodof Froehler, B. C., et al., Nucleic Acids Res (1986) 14:5399-5407.

EXAMPLE 2 Preparation of Nucleotides and Oligomers

2′-S-phenylcytidine was prepared from2,2′-anhydro-(1-B-D-arabinofuranosyl) cytosine-HCl (a cyclic nucleoside)by suitable treatment with thiophenol. The —NH₂ of cytosine and5′-hydroxy were protected and the 3′ —OH activated as follows:

2′-S-Phenylthiocytidine. To a solution of 500 mg (1.91 mmol) of2,2′-anhydro-(1-B-D-arabinofuranosyl)-cytosine hydrochloride (purchasedfrom Sigma) in 50 ml of dry DMF and 1.86 ml of dry TEA (13.3 mmol) wasadded 0.980 ml (9.54 mmol, 5.0 equiv) of thiphenol. The reaction wasstirred for 5 h and then concentrated. The residue was concentrated fromMeOH onto 2.00 g of silica gel. The powder was added to the top of a 30mm column of silica gel that was equilibrated with CH₂Cl₂. The columnwas then eluted with one column volume of CH₂Cl₂, then one column volumeof 6.25% MeOH in CH₂Cl₂, then one column volume of 12.5% MeOH in CH₂Cl₂and then 25% MeOH in CH₂Cl₂. Concentration of the product fractionsafforded 529 mg (82.5% yield) of product as a near colorless oil.

N⁴-Benzoyl-2′-S-pheylthiocytidine. The method of transient protection(Ti, G. S., et al., J Am Chem Soc (1982) 104:1316-1319) was used toprepare the title compound. To 429 mg (1.28 mmol) of2′-S-phenylthiocytidine (first concentrated from dry pyridine) in 12.2ml of dry pyridine that was cooled on an ice-water bath was added 0.832ml (6.56 mmol) of chlorotimethylsilane. The reaction was stirred for 15min, and then 0.767 ml (6.61 mmol) of benzoyl chloride was added. Theice bath was removed and stirring continued for 2.5 h. The reaction wasagain cooled on an ice-water bath, and 2.56 ml of H₂O added. Thereaction was stirred for 5 min, and then 2.56 ml of concentrated aqueousNH₄OH was added. Stirring was continued for 30 min, and then the mixturewas partitioned between EtOAc and H₂O, shaken and separated. The aqueouslayer was extracted with EtOAc, and the combined organics were washedwith H₂O, dried (Na₂SO₄), filtered, and concentrated. The, residue waspurified by flash chromatography on a 30 mm column using one columnvolume of CH₂Cl₂ and then 5% MeOH in CH₂Cl₂ as eluants. Concentration ofthe product fractions afforded 138 mg (32.2% yield) of product as anoil.

5′-O-(4,4′-Dimethoxytrityl)-2′-S-phenylthio cytidine. To 138 mg (0.314mmol) of N⁴-benzoyl-2′-S-phenylthiocytidine (that was first concentratedfrom dry pyridine) in 2.00 ml of dry pyridine was added 128 mg (0.377mmol, 1.2 equiv) of 4,4′-dimethoxytritylchloride. The reaction wasstirred for 18 h at room temperature and then diluted with 2.00 ml ofH₂O. The mixture was partitioned between Et₂O and H₂O, shaken andseparated. The aqueous layer was extracted with Et₂O, and the combinedorganics washed with 1% aqueous NaHCO₃, dried (Na₂so₄), filtered, andconcentrated. The crude product was purified by flash chromatography ona 30 mm column using one column volume of CH₂Cl₂ then 2.5% MeOH inCH₂Cl₂, and then 5% MeOH in CH₂Cl₂ as eluants. Concentration of theproduct fractions afforded 206 mg (88.4% yield) of product.

5′-O-(4,4′-Dimethyoxytrityl)-2′-S-phenylthiocytidin-3′-yl-hydrogenphosphonatetriethylammonium salt. The preparation of this hydrogenphosphonate wasthe same as that described above except that 206 mg (0.278 mmol) of5′-O-(4,4′-dimethoxytrityl)-2′-S-phenylthiocytidine was used, and thereagents were adjusted to the 0.278 mmol scale. After the TEAB workup,the organics were washed with 1 M aqueous TEAB, dried (Na₂SO₄),filtered, and concentrated. The residue was purified on a 30 mm columnusing one column volume of 2% TEA in CH₂Cl₂, then 2% TEA and 5% MeOH inCH₂Cl₂, and then 2% TEA and 10% MeOH in CH₂Cl₂ as eluants. The productfractions were concentrated to a foam which was partitioned betweenCH₂Cl₂ and 1 M aqueous TEAB, shaken, and separated. The organic layerwas dried (Na₂SO₄), filtered, and concentrated. The product wasconcentrated from dry CH₃CN affording 165 mg (65.5% yield) of product asa foam.

The resulting title compound is coupled into oligomers using the methodof Froehler, B. C., et al., Nucleic Acids Res (1986) 14:5399-5407.

EXAMPLE 3 Preparation of Additional Nucleotides and Oligomers

A. (A=OCH₂COOEt)

The 2′-derivatized nucleoside was prepared fromN⁴-benzoyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine byreaction with ethyl iodoacetate and deprotection of the 2′ and 5′hydroxyls. The 5′ position was protected and the 3′ position convertedto a group reactive to link hydroxyl groups.

N⁴-Benzoyl-2′-O-(ethoxycaronylmethyl)-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine. The preparation ofthis compound was an adaptation of a similar reaction used for thepreparation of the 2′-OMe (Inoue, H., et al., Nucleic Acids Res (1987)15:6131-6149). To 250 mg ofN⁴-benzoyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine(Markiewicz, W. J., J Chem Res (1979) 181-196) (0.424 mmol; firstconcentrated from benzene) was added 1.00 ml (8.45 mmol, 19.9 equiv) ofethyl iodoacetate, followed by 294 mg of Ag₂O (1.27 mmol, 2.99 equiv).The mixture was rapidly stirred and heated at 42° C. for 32 h. Themixture was then filtered and concentrated. The residue was taken up inCH₂Cl₂, treated with H₂S, and concentrated. The dark residue waspurified by flash chromatography on a 25 mm column using one columnvolume of CH₂Cl₂, then one column volume of 2.5% MeOH in CH₂Cl₂, and the5% MeOH in CH₂Cl₂ as eluants. Concentration of the product fractionsafforded 270 mg (94.4% yield) of product as a foam.

N⁴-Benzoyl-2′-O-(ethoxycarbonylmethyl)-cytidine. To 170 mg of N⁴-benzoyl-2′-O-(ethoxycarbonylmethyl)-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine(0.252 mmol) in 1.70 ml THF was added 0.126 ml of a 1.0 M solution oftetrabutylammonium fluoride in THP (0.126 mmol, 0.50 equiv). Thereaction was stirred at room temperature for 70 min. Then 3.4 ml ofpyridine/EtOH/H₂O 3/1/1 (v/v/v) was added and stirring continued for 5min. Then approximately 10 ml of Amberlyst A-21 ion-exchange resin(pyridinium form) was added and stirring continued for 5 min. Themixture was filtered, and the resin rinsed with EtOH. The combinedfiltrates were concentrated. The residue was then concentrated from EtOHonto 1.00 g of silica gel. This powder was loaded onto the top of a 25mm column of silica gel that had been equilibrated with CH₂Cl₂. Thecolumn was eluted with one column volume of CH₂Cl₂, then one columnvolume of 2.5% MeOH in CH₂Cl₂, and then 5% MeOH in CH₂Cl₂. Concentrationof the product fractions afforded 40.7 mg (37.6% yield) of product as anoil.

N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-(ethoxycarbonylmethyl)-cytidine.To 40.7 mg of N⁴-benzoyl-2′-O-(ethoxycarbonylmethyl)-cytidine (0.0939mmol, first concentrated from dry pyridine) in 1.00 ml dry pyridine wasadded 38.3 mg of 4,4′-dimethoxytritylchloride (0.113 mmol, 1.2 equiv).The reaction was stirred at room temperature for 20 h and then dilutedwith 0.50 ml of H₂O. The mixture was partitioned between Et₂O and H₂O,shaken and separated. The aqueous layer was extracted with Et₂O. Thecombined organics were washed with 1% aqueous NaHCO₃, dried (Na₂SO₄),filtered, and concentrated. The residue was purified by flashchromatography on a 20 mm column using one column volume CH₂Cl₂, then2.5% MeOH in CH₂Cl₂, and then 5.0% MeOH on CH₂Cl₂ as eluants.Concentration of the product fractions afforded 55.2 mg (79.9% yield) ofproduct.

N⁴-Benzoyl-5′-O-(5,5′-dimethoxytrityl)-2′-O-(ethoxycarbonylmethyl)-cytidin-3′-yl-hydrogenphosphonatetriethylammonium salt. The preparation of this compound was the same asdescribed in the earlier preparation of hydrogenphosphates except thatin this case 55.2 mg (0.0750 mmol) ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-(ethoxycarbonylmethyl)-cytidinewas used, and the reagents adjusted for the 0.0750 mmol scale. After theTEAB workup, the organic layer was washed with 1 M aqueous TEAB, dried(Na₂SO₄), filtered, and concentrated. The residue was purified by flashchromatography on a 25 mm column using one column volume of 1% TEA inCH₃CN, then one column volume of 1% TEA and 5% H₂O in CH₃CN, and then 1%TEA and 10% H₂O in CH₃CN. Concentration of the product fractionsafforded a foam which was partitioned between CH₂Cl₂ and 1 M aqueousTEAB, shaken, and separated. The organics were dried (Na₂SO₄), filtered,and concentrated. The product was concentrated from dry CH₃CN affording23.8 mg (35.2% yield) of product.

The derivatized resulting compound of the previous paragraph wasincluded in an oligomer as described above.

B. (A=OEt)

N⁴-Benzoyl-2′-O-ethyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidinewas prepared similarly toN⁴-benzoyl-2′-O-(ethoxycarbonylmethyl)-3′,5′-O-(tetraisoproyldisiloxane-1,3-diyl)-cytidineexcept that iodoethane was used in place of ethyl iodoacetate. The titlecompound was then converted toN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-ethyl-cytidin-3′-yl-hydrogenphosphonatetriethylammonium salt using the same sequence of steps as for the2′-O-(ethoxycarbonylmethyl)-compound, and further included in oligomersas described above.

C. (A=OCH₂CH₂CH₂CH₃)

N⁴-Benzoyl-2′-O-butyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidinewas prepared similarly toN⁴-benzoyl-2′-O-(ethoxybonylmethyl)-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidineexcept that iodobutane was used in place of ethyl iodoacetate. The titlecompound was then converted toN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-butyl-cytidin-3′-yl-hydrogenphosphonatetriethylammonium salt using the same sequence of steps as for the2′-O-(ethoxycarbonylmethyl)-compound, and further included in oligomersas described above.

D. (A=O—SiMe2tBu)

5′-O-(4,4′-Dimethoxytrityl)-2′-O-t-butyldimethylsilyluridin-3′-yl-hydrogenphosphonateDBU salt. This compound was prepared differently than described in theliterature. The preparation of this compound was the same as for theabove compound except that 400 mg (0.606 mmol) of5′-O-(4,4′-dimethoxytrityl)-2′-O-t-butyldimethylsilyluridine (purchasedfrom Peninsula Labs) was used and the rest of the conditions were scaledto the 0.606 mmol scale. After the TEAB workup, the organic layer wasdried (Na₂SO₄), filtered and concentrated. The residue was purified byflash chromatography on a 35 mm column using one column volume of 1% TEAin CH₂Cl₂, then one column volume of 1% TEA and 4% MeOH in CH₂Cl₂, andthen 1% TEA and 8% MeOH in CH₂Cl₂ as eluants. The product was isolatedand concentrated. The foam was partitioned between CH₂Cl₂ and 1 Maqueous 1,8-diazabicyclo[5.4.0]undec-7-ene bicarbonate (DBU bicarbonate,pH=9.0), shaken and separated. The organic layer was again washed with50 mL of 1 M aqueous DBU bicarbonate, dried (Na₂SO₄), filtered andconcentrated. The product was concentrated from dry CH₃CN affording 435mg (81.9% yield) of product as an oil. The resulting derivative isincluded in an oligomer synthesized by standard methods.

EXAMPLE 4 Conversion of 2′ Substituents in Oligomers

N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-(ethoxycarbonylmethyl)-cytidin-3′-yl-hydrogenphosphonatetriethylammonium salt was coupled into oligonucleotides using thehydrogen phosphonate method.

In order to generate the 2′-OCH₂CO₂H, the oligo was deprotected, cleavedfrom the support, and the 2′-OCH₂CO₂Et hydrolyzed to the 2′-OCH₂CO₂Husing 0.1 M aqueous NaOH at 45° C. for 4.5 h.

In order to generate the 2′-OCH₂CONH₂, the above oligo containing the2′-OCH₂CO₂Et was deprotected, cleaved from the support and the2′-OCH₂CO₂Et converted to the 2′-OCH₂CONH₂ using NH₃ in MeOH at 45° C.for 29 h.; the corresponding amide of the formula —OCH₂CONHCH₂CH₂NH₂ wasprepared similarly.

EXAMPLE 5 Resistance to Nuclease Activity of the Compounds of theInvention

The ability of dimers of the compounds synthesized in Example 1 toresist the activity of nucleases was determined. The illustratedcompounds of Formula 1 were coupled to an additional unmodifiedthymidine using standard procedures to obtain compounds of the formula:

The resulting dimeric compounds were tested for stability with respectto nuclease from snake venom as described above. The following resultsare shown in Table 1.

TABLE 1 Snake Venom % dimer after Time for 100% —X—Y—Z 5 min.degradation —H  0% <5 min. —OCH₃  20% — —SPh  75% >140 min. —OCH₂CO₂H 40% 46 min. —OCH₂CONH₂  51% >42 min. —OTBS >99% >>140 min. (77% dimerafter 140 min.) —OCH₂CONHCH₂CH₂NH₂ 100% >>140 min. (>99% dimer after 140min.

As shown in Table 1, the addition of the substituent to the 2′-positiongreatly enhances the stability of the resulting dimer to nucleasecleavage.

1. A mixed sequence oligonucleotide having the formula:

or a salt thereof, wherein: each B independently is a protected orunprotected purine or pyrimidine base in the β-anomeric configuration;W₃ and W₄ are each independently H, PO₃ ⁻², a protecting group, or anintermediate moiety in the formation of an internucleotide link whichwhen reacted with the appropriate —OH results in Z; n is an integer of1-200; each Z is independently P(O)O, P(O)S or P(O)N(R)₂; each R isindependently H or alkyl (C₁₋₆); each A is H,OH, O—Pr wherein Pr is aprotecting group, or X-Y wherein X-Y is O-(unsubstituted alkyl (C₂₋₂₀)),S-(unsubstituted alkyl (C₁₋₂₀)), or NH-(unsubstituted alkyl (C₁₋₂₀));and at least one A is X-Y.
 2. The oligonucleotide of claim 1 wherein W₃and W₄ are H.
 3. The oligonucleotide of claim 1 wherein at least one W₃or W₄ is a group reactive to link hydroxyl groups.
 4. Theoligonucleotide of claim 1 wherein at least one Z is P(O)S or P(O)N(R)₂.5. The oligonucleotide of claim 1 wherein at least one Z is P(O)N(R)₂.6. The oligonucleotide of claim 1 wherein at least one Z is P(O)S. 7.The oligonucleotide of claim 1 wherein X-Y is O-(unsubstituted alkyl(C₂₋₆)), S-(unsubstituted alkyl (C₂₋₆)), or NH-(unsubstituted alkyl(C₂₋₆)).
 8. The oligonucleotide of claim 1 wherein B is a conventionalpurine or pyrimidine.
 9. The oligonucleotide of claim 8 wherein B isadenine, thymine, cytosine, guanine or uracil.
 10. The oligonucleotideof claim 1 wherein at least one A is H.
 11. The oligonucleotide of claim1 wherein at least one A is OH.
 12. The oligonucleotide of claim 1wherein X-Y is O-(unsubstituted alkyl (C₂₋₆)).
 13. The oligonucleotideof claim 1 wherein X-Y is S-(unsubstituted alkyl (C₂₋₆)).
 14. Theoligonucleotide of claim 1 wherein X-Y is NH-(unsubstituted alkyl(C₂₋₆)).